1. Field of the Invention
The present invention is concerned with novel peptides which inhibit renin.
The present invention is also concerned with pharmaceutical compositions containing the novel peptides of the present invention as active ingredients, with methods of treating renin-associated hypertension and hyperaldosteronism, with diagnostic methods which utilize the novel peptides of the present invention, and with methods of preparing the novel peptides of the present invention.
Renin is a proteolytic enzyme of molecular weight about 40,000, produced and secreted by the kidney. It is secreted by the juxtaglomerular cells and acts on the plasma substrate, angiotensinogen, to split off the decapeptide angiotensin I, which is converted to the potent pressor agent angiotensin II. Thus, the renin-angiotensin system plays an important role in normal cardiovascular homeostasis and in some forms of hypertension.
In the past, attempts to modulate or manipulate the renin-angiotensin system have met with success in the use of inhibitors of angiotensin I converting enzyme. In view of this success, it seems reasonable to conclude that a specific inhibitor of the limiting enzymatic step that ultimately regulates angiotensin II production, the action of renin on its substrate, would be at least equally successful. Thus, an effective inhibitor of renin has been long sought as both a therapeutic agent and as an investigative tool.
2. Brief Description of the Prior Art
There has been substantial interest in the synthesis of useful renin inhibitors for many decades; and the following table lists the major classes of renin inhibitors that have been studied, as well as their inhibition constants (K.sub.i):
______________________________________ Class K.sub.i (M) ______________________________________ Renin antibody probably 10.sup.-6 Pepstatin 10.sup.-6 - 10.sup.-7 Phospholipids 10.sup.-3 Substrate analogs Tetrapeptides 10.sup.-3 Octa- to tridecapeptides 10.sup.-5 - 10.sup.-6 ______________________________________
Umezawa et al., in J. Antibiot. (Tokyo) 23: 259-262, 1970, reported the isolation of a peptide from actinomyces that was an inhibitor of aspartyl proteases such as pepsin, cathepsin D, and renin. This peptide, known as pepstatin, was found by Gross et al., Science 175:656, 1971, to reduce blood pressure in vivo after the injection of hog renin into nephrectomized rats. However, pepstatin has not found wide application as an experimental agent because of its limited solubility and its inhibition of a variety of other acid proteases in addition to renin. The structure of pepstatin is shown below: ##STR2##
To date, many efforts have been made to prepare a specific renin inhibitor based on substrate analogy. Since the human renin substrate has only recently been elucidated (Tewksbury et al., Circulation 59, 60, Supp. II: 132, Oct. 1979), heretofore substrate analogy has been based on the known pig renin substrate. While the human and pig renin substrates are not the same, the substrate analogy based on pig renin has always been considered acceptable in the art as predictive of human renin inhibitory activity because of the closely related activity of the two renins. Thus, while pig renin does not cleave the human renin substrate, human renin, on the other hand, does cleave the pig renin substrate. See Poulsen et al., Biochim. Biophys. Acta 452:533-537, 1976; and Skeggs, Jr. et al., J. Exp. Med. 106:439-453, 1957.
It has been found, for example, using pig renin substrate analogy, that the octapeptide sequence extending from histidine-6 through tyrosine-13 has kinetic parameters essentially the same as those of the full tetradecapeptide renin substrate. The amino acid sequence of the octapeptide in pig renin substrate is as follows: ##STR3## Renin cleaves this substrate between Leu.sup.10 and Leu.sup.11.
The renin inhibitory peptides of the present invention are based on human renin substrate analogy. The human renin octapeptide sequence, related to the pig renin substrate octapeptide, is as follows: ##STR4## Similar to the pig renin substrate, human renin cleaves this substrate between Leu.sup.10 and Val.sup.11.
Kokubu et al., Biochem. Pharmacol. 22: 3217-3223, 1973, synthesized a number of analogs of the tetrapeptide found between residues 10 to 13 of the pig renin substrate, but while inhibition could be shown, inhibitory constants were only of the order of 10.sup.-3 M.
Analogs of a larger segment of renin substrate were also synthesized: Burton et al., Biochemistry 14: 3892-3898, 1975, and Poulsen et al., Biochemistry 12: 3877-3882, 1973. Two of the major obstacles which had to be overcome to obtain an effective renin inhibitor useful in vivo were lack of solubility and weak binding (large inhibitory constant). Modifications to increase solubility soon established that the inhibitory properties of the peptides are markedly dependent on the hydrophobicity of various amino acid residues, and that increasing solubility by replacing lipophilic amino acids with hydrophilic isosteric residues becomes counter-productive. Other approaches to increasing solubility have had limited success. Various modifications designed to increase binding to renin have also been made, but here too, with only limited success. For a more detailed description of past efforts to prepare an effective inhibitor of renin, see Haber and Burton, Fed. Proc. Fed. Am. Soc. Exp. Biol. 38:2768-2773, 1979.
For other articles describing previous efforts to devise renin inhibitors, see Marshall, Federation Proc. 35: 2494-2501, 1976; Burton et al., Proc. Natl. Acad. Sci. USA 77: 5476-5479, Sept. 1980; Suketa et al., Biochemistry 14: 3188, 1975; Swales, Pharmac. Ther. 7: 173-201, 1979; Kokubu et al., Nature 217: 456-457, Feb. 3, 1968; Matsushita et al., J. Antibiotics 28: 1016-1018, Dec. 1975; Lazar et al., Biochem. Pharma. 23: 2776-2778, 1974; Miller et al., Biohem. Pharma. 21: 2941-2944, 1972; Haber, Clinical Science 59: 7s-19s, 1980; and Rich et al., J. Org. Chem. 43: 3624, 1978, and J. Med. Chem. 23: 27, 1980.